PROJECT SUMMARY/ABSTRACT Targeted rewriting of the genetic code of developed cells for therapeutic benefit is a longstanding goal in the biomedical sciences. Base editing is a new genome engineering approach that harnesses the sequence- specific targeting ability of a catalytically inactive or impaired Cas9 nuclease to target nucleobase-tailoring enzymes to specific locations in the genome, eventually causing targeted single base mutations. In the ternary complex between Cas9, sgRNA, and the target DNA, a portion of the non-templated strand of DNA forms an exposed, disordered, single-stranded loop. In base editing, an enzyme fused to Cas9 takes advantage of this opportunity and catalyzes chemical modifications on nucleobases within a small segment of this loop. These modifications are designed to alter the base pairing properties of the modified nucleotide, and miscoding during DNA replication or repair effects the eventual point mutation. The long term goal of this project is to discover productive base pairing interactions of modified nucleobases and to harness these modifications in developing base editors capable of selectively effecting every permutation of a point mutation. A protein engineering approach to optimize new nucleobase-oxidizing enzymes will be undertaken, and a combination of chemical synthesis and enzymology will allow for the evaluation of the miscoding potential of modified nucleobases. Base editors are anticipated to have a direct impact on human health: the majority of pathogenic human genetic mutations are point mutations, and base editing has the potential to reverse these mutations in somatic cells.